Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.
Introduction Colocalization analysis has become a common tool of microscopic subcellular localization studies. There are many tools and packages available for colocalization analyses. MINER2.0 differs from many other packages by providing user‐friendly, highly customizable and scalable Nearest Neighbour analyses, drawing on principles of centroid localization used in single molecule imaging. Methods and Results To circumvent issues related to colocalization indices that rely on (often) arbitrarily thresholding color channels, our analysis pipeline compares predefined sets of regions of interest (ROIs) or can perform a de novo search for the K‐Nearest Neighbours (KNN) around the predefined ROIs. The algorithm calculates and reports: puncta area, mean intensities, full‐width‐half‐max sizes and intensity, and distances between the centroid or perimeter of reference ROIs and the centers of ROIs in up to two comparator ROI sets. Users can define ROI centroids as center of mass, geometric center or the biaxial Gaussian fit of the puncta. This latter method can statistically detect differences in distances between centroids in separate image channels of 1.4 pixels. At higher magnifications this allows detection of distances separating centroids in separate color channels at <90 nm, below the diffraction limit of optical microscopy. The pipeline is written as a user‐friendly macro for ImageJ/Fiji to ease of access. New in version 2.0, we moved from a brute force KNN calculation in ImageJ with an O(n2) time complexity to a Kd‐tree calculation in R with O(nLog(n)) complexity. This reduces the KNN calculation for large ROI sets (i.e. >1000 ROIs) from hours in ImageJ to typically <10 s in R. This enables batched analysis of large image sets on standard desktop computers (e.g. 36,000 ROIs in six 2048 × 2048 pixel images analyzed in <1.5 hours). We discuss three use cases: (1) the analysis of anti‐colocalization of the vesicular nucleotide transporter with diverse vesicle and lysosomal markers in Neuro2A cells, (2) analysis of the decrease of mitochondria containing mitochondrial DNA in A7r5 cells treated with diverse stressors including angiotensin II, 5,6‐dideoxycytidine, rotenone and hydrogen peroxide, and (3) the prevalence of micronuclei as markers of genotoxic stress in A7r5 cells expressing loss‐of‐function variants of polymerase gamma that is responsible for replicating mitochondrial DNA. Conclusion MINER2.0 provides a user‐friendly interface for fast batch processing of spatial colocalization analyses with minimal subjective bias. It provides users with extensive output parameters to allow detailed understanding of the spatial relationships of fluorescently labelled structures within cells. Support or Funding Information Natural Sciences and Engineering Research Council of Canada
We investigated the role of mitochondria (MT) in calcium signaling in a culture of rat aortic smooth muscle cells. We used targeted aequorin to selectively measure [Ca2+] in this organelle. Our results reveal that smooth muscle cell stimulation with agonists causes a large, transient increase in mitochondrial [Ca2+] ([Ca2+]m). This large transient can be blocked with inhibitors of the sarco-endoplasmic reticulum Ca2+-ATPase, suggesting a close relationship between the sarcoplasmic reticulum (SR) and the mitochondria. FCCP completely abolished the response to agonists, and targeted mitochondrial GFP revealed a vast tubular network of MT in these cells. When added before stimulation with ATP, IP3 inhibitors partially blocked the ATP-induced rise in mitochondrial Ca2+ release. The role of the Na+/Ca2+ exchanger (NCX) was examined by removing extracellular Na+. This procedure prevented the decrease in the [Ca2+]m transient normally seen on removal of extracellular Ca2+. We propose a functional linkage of MT and SR dependent on a narrow junctional space between the two organelles in which Ca2+ diffusion is restricted. Approximately half of the mitochondria appear to be associated with the superficial SR, which communicates with the extracellular space via NCX.
Subplasmalemmal ion fluxes have global effects on Ca 2+ signaling in vascular smooth muscle. Measuring cytoplasmic and mitochondrial [Ca 2+ ]and [Na + ], we previously showed that mitochondria buffer both subplasmalemmal cytosolic [Ca 2+ ] and [Na + ] in vascular smooth muscle cells. We have now directly measured sarcoplasmic reticulum [Ca 2+ ] in aortic smooth muscle cells, revealing that mitochondrial Na + /Ca 2+ exchanger inhibition with CGP-37157 impairs sarcoplasmic reticulum Ca 2+ refilling during purinergic stimulation. By overexpressing hFis1 to remove mitochondria from the subplasmalemmal space, we show that the rate and extent of sarcoplasmic reticulum refilling is augmented by a subpopulation of peripheral mitochondria. In ATP-stimulated cells, hFis-1–mediated relocalization of mitochondria impaired the sarcoplasmic reticulum refilling process and reduced mitochondrial [Ca 2+ ] elevations, despite increased cytosolic [Ca 2+ ] elevations. Reversal of plasmalemmal Na + /Ca 2+ exchange was the primary Ca 2+ entry mechanism following ATP stimulation, based on the effects of KB-R7943. We propose that subplasmalemmal mitochondria ensure efficient sarcoplasmic reticulum refilling by cooperating with the plasmalemmal Na + /Ca 2+ exchanger to funnel Ca 2+ into the sarcoplasmic reticulum and minimize cytosolic [Ca 2+ ] elevations that might otherwise contribute to hypertensive or proliferative vasculopathies.
